Purpose: To test ribozymes targeting mouse telomerase RNA (mTER) for suppression of the progression of B16-F10 murine melanoma metastases in vivo.

Experimental Design: Hammerhead ribozymes were designed to target mTER. The ribozyme sequences were cloned into a plasmid expression vector containing EBV genomic elements that substantially prolong expression of genes delivered in vivo. The activity of various antitelomerase ribozymes or control constructs was examined after i.v. injection of cationic liposome:DNA complexes containing control or ribozyme constructs. Expression of ribozymes and mTER at various time points were evaluated by quantitative real-time PCR. Telomerase activity was examined using the telomeric repeat amplification protocol.

Results: Systemic administration of cationic liposome:DNA complexes containing a plasmid-expressed ribozyme specifically targeting a cleavage site at mTER nucleotide 180 significantly reduced the metastatic progression of B16-F10 murine melanoma. The antitumor activity of the anti-TER 180 ribozyme in mice was abolished by a single inactivating base mutation in the ribozyme catalytic core. The EBV-based expression plasmid produced sustained levels of ribozyme expression for the full duration of the antitumor studies. In addition to antitumor activity, cationic liposome:DNA complex-based ribozyme treatment also produced reductions in both TER levels and telomerase enzymatic activity in tumor-bearing mice.

Conclusions: Systemic, plasmid-based ribozymes specifically targeting TER can reduce both telomerase activity and metastatic progression in tumor-bearing hosts. The work reported here demonstrates the potential utility of plasmid-based anti-TER ribozymes in the therapy of melanoma metastasis.

Telomeres are complexes at the termini of eukaryotic chromosomes that play a crucial role in maintaining chromosomal integrity (1, 2). In many normal cells, telomeric DNA shortens progressively with each cell division, and upon reaching a critically short length, it can trigger a state of replicative senescence (3). At this point, the cells may be susceptible to apoptotic cell death. In contrast, cancer cells escape senescence through both cell cycle checkpoint inactivation and the activation of telomerase. Telomerase contains essential RNA [Ref. 3; telomerase RNA (TER)] and protein [telomerase reverse transcriptase (TERT)] components (4, 5). The importance of telomerase in cancer is supported by studies showing that telomerase expression promotes production of tumorigenic cell lines (6, 7) and by its very frequent activation in human cancers (8, 9, 10). However, to date, no study has examined the effects of targeting telomerase on metastatic progression in tumor-bearing hosts.

Based on the significant role that telomerase has been shown to play in tumor cell proliferation, there has been considerable interest in developing therapeutic strategies aimed at targeting telomerase in cancer cells (11). These strategies have included antisense- or ribozyme-targeting of telomerase (12, 13) and using dominant-negative TERT (14) or mutant TERs (15). Studies using antisense constructs directed against TER or TERT have shown induction of tumor cell apoptosis in vitro(12, 13, 16). Reduced tumor cell growth in vivo was shown in studies using dominant-negative TERT (14) and mutant TERs (15). However, in prior studies using ribozymes to target various components of the telomerase complex, effects on tumor cell proliferation were only measured in vitro(13, 17, 18, 19). Therefore, in this study, we investigated the antitumor activity of systemically delivered, plasmid-based hammerhead ribozymes targeting telomerase in a murine model of melanoma metastasis.

Plasmid Construction, Cloning, and Preparation.

DNA encoding the ribozymes listed below was cloned into the EBV-based expression plasmid as described previously (20, 21, 22).

Anti-TER ribozyme sequences are as follows: 5′-ACA AAA CTG ATG AGT CCG TGA GGA CGA AAC CAG AA-3′ (nucleotide 79); 5′-GAG AAA CTG ATG AGT CCG TGA GGA CGA AAC AGC GG-3′ (nucleotide 100); 5′-AGG TAA CTG ATG AGT CCG TGA GGA CGA AAC ACC GA-3′ (nucleotide 405); and 5′-TGC GCT CTG ATG AGT CCG TGA GGA CGA AAC GTT TG-3′ (nucleotide 180). The anti-TER 180 mutant ribozyme sequence was also generated with a single base change (G to C, in bold) in the ribozyme catalytic core, with the following sequence: 5′-TGC GCT CTC ATG AGT CCG TGA GGA CGA AAC GTT TG-3′.

Cell Culture and Tumor Inoculation.

Murine B16-F10 melanoma cells (American Type Culture Collection, Manassas, VA) were maintained in culture as described previously (21, 22).

Preparation of Cationic Liposomes and Cationic Liposome:DNA Complexes.

Liposome (100 mm), containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, was prepared as described previously (21). For in vivo studies, 25 μg of plasmid DNA and 650 nmol of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (26 nmol lipid/μg DNA) were prepared and injected via tail vein into each mouse as described previously (21, 22). This ratio of cationic lipid to DNA has been previously shown to produce efficient gene transfer in vivo and evidence of biological activity in a number of different in vivo model systems (20, 21, 22).

Animal Studies.

Groups of ten 45-day-old female C57Bl/6 mice (Charles River, Willmington, MA) were i.v. inoculated with 3 × 104 B16-F10 melanoma cells (day 0) and 200 μl of cationic liposome:DNA complex containing control or therapeutic genes (days 3 and 10) as described previously (21, 22). For samples analyzed by telomeric repeat amplification protocol, cationic liposome:DNA complexes containing the same control or ribozyme inserts were re-injected into mice at 24, 48, or 72 h before the mice were sacrificed. For tumor counts and telomeric repeat amplification protocol, mice were euthanized by halothane inhalation by experiment day 30, and the lungs dissected out. To analyze the time course of ribozyme expression by quantitative real-time PCR, mice were euthanized as above on sequential days, and the lungs were dissected out. For tumor counts, the pulmonary metastases were counted in a blinded fashion as described previously (21, 22). For telomeric repeat amplification protocol, individual tumors were resected off mouse lungs, quick-frozen on dry ice, and processed for telomeric repeat amplification protocol activity and total protein quantification. For TaqMan, the mouse lungs were preserved in RNAlater (Ambion, Austin, TX) and processed for RNA analyses.

Quantitative Real-Time PCR Analysis.

Total RNA was extracted from the lungs of tumor-bearing mice using the RNeasy Maxi Kit (Qiagen). For quantitative real-time PCR (TaqMan assay), cDNA was synthesized from 250 ng of DNase I-digested total cellular RNA, using a final reaction concentration of 1× GeneAmp PCR Buffer II (Applied Biosystems, Foster City, CA), 7.5 mm MgCl2 solution (Applied Biosystems), 1 mm each dNTP (Invitrogen), 5 μm random primers (Invitrogen), 0.4 unit/μl RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and 0.6 unit/μl Superscript II. The reaction was incubated at 25°C for 10 min, 48°C for 50 min, and 70°C for 15 min. To quantitate mTER expression levels, cDNA was synthesized from DNase I-digested total cellular RNA using iScript cDNA Synthesis kit (BioRad Laboratories, Hercules, CA). For TaqMan analysis, 5 μl of cDNA was amplified using a final concentrations of 500 nm (each) forward and reverse primers, 200 nm probe (Integrated DNA Technologies, Inc.), 200 μm (each) dNTPs (Invitrogen), 1× buffer A (Applied Biosystems), and 0.025 unit/μl AmpliTaq Gold DNA Polymerase (Applied Biosystems). The amplification was conducted at 95°C for 12 min and 45 cycles of 95°C for 15 s and 60°C for 1 min. The TaqMan probe and primer sequences were as follows:

Mammalian histone control gene: forward primer, 5′-GCT TCC AGA GCG CAG CTA TC-3′; reverse primer, 5′-GGC GTG CTA GCT GGA TGT CT-3′; TaqMan probe, 5′-FAM-TGC TTT GCA GGA GGC AAG TGA GGC-TAMRA-3′.

Mouse gus control gene: forward primer, 5′-CTC ATC TGG AAT TTC GCC GA-3′; reverse primer, 5′-GGC GAG TGA AGA TCC CCT TC-3′; TaqMan probe, 5′-FAM- CGA ACC AGT CAC CGC TGA GAG TAA TCG -TAMRA-3′.

Ribozyme (forward primer targets the ribozyme constant region): forward primer, 5′-CTG ATG AGT CCG TGA GGA CGA-3′; reverse primer, 5′-GTA ATT TGT CCT CCA GAT CGC AG-3′; TaqMan probe, 5′-FAM-AGG TGG GCG GGC CAA GAT AGG G-TAMRA-3′.

Mouse TER: forward primer, 5′-GTCTTTTGTTCTCCGCCCG-3′; reverse primer, 5′-CGGCGAACCTGGAGCTC-3′; TaqMan Probe, 5′-FAM-CGTTCCCGAGCCTCAAAAACAAACG-TAMRA-3′.

For the probe and primers of each gene, a high PCR efficiency (greater than 90%) was ascertained before the expression analyses. Also, reverse transcriptase linearity tests revealed linear reverse transcription with 250–500 ng total cellular RNA/reverse transcriptase reaction. For each sample, run in triplicate, expression levels of the gene of interest (the ribozyme or mTER) and control genes (Gus or Histone) were determined by quantitative real-time PCR, with parallel analysis of no reverse transcriptase controls to rule out DNA contamination. For each sample, the cycle threshold of the gene of interest (ribozyme or mTER) was normalized against the cycle thresholds of histone or Gus control genes, before determination of relative gene expression levels between samples.

Telomeric Repeat Amplification Protocol Assays.

Telomeric repeat amplification protocol assays were performed according to the manufacturer’s TRAPeze protocol, using end-labeled primers (TRAPeze Telomerase Detection kit; Intergen Co., Purchase, NY).

Four hammerhead ribozymes were designed to target cleavage sites in murine TER (Ref. 23; Fig. 1,A). Oligonucleotides encoding the ribozymes were cloned into an expression plasmid containing the human cytomegalovirus promoter and EBV-based elements (EBV nuclear antigen-1 and family of repeats; Ref. 20; Fig. 1 B) and verified by sequence analysis. The transfection efficiency of cationic liposome:DNA complexes encoding the anti-TER ribozymes was assessed using luciferase reporter gene cotransfection in vitro (data not shown). Expression of the anti-TER ribozymes was detectable by reverse transcriptase-PCR analysis of total cellular RNA extracted both after in vitro transfection of B16 cells and from lungs of C57Bl/6 mice bearing metastatic syngeneic B16-F10 melanoma tumors (data not shown).

Because the lungs of tumor-bearing mice were shown to express the delivered anti-mTER ribozymes, the potential antitumor activity of these anti-mTER ribozymes was subsequently investigated in C57Bl/6 mice bearing B16-F10 tumors. Groups of 10 C57Bl/6 mice received i.v. injections of B16-F10 cells and then subsequently treated i.v. with cationic liposome:DNA complexes containing an anti-mTER ribozyme or control sequence 3 and 10 days after tumor cell injection. Two of the anti-TER ribozymes (those targeting mTER nucleotides 180 and 405) reduced metastatic burden (P < 0.05, Fig. 2) at statistically significant levels. The other two anti-mTER ribozymes tested suppressed melanoma progression to some extent, as evidenced by the reduced number of melanotic lung tumors (Fig. 2). In addition, we performed preliminary investigations into the antitumor potential of three ribozymes targeting TERT mRNA. Plasmid-based expression of the anti-TERT ribozymes was also verified by reverse transcriptase-PCR analysis (data not shown). None of the three anti-TERT ribozymes tested to date produced significant antimetastatic activity when using the same protocol of systemic cationic liposome:DNA complex delivery into tumor-bearing C57Bl/6 mice (data not shown). The anti-TER 180 ribozyme targets the pseudoknot domain of mTER (23). Based on the above results, and on the importance of the pseudoknot domain for telomerase activity (24) and its evolutionary conservation (23), the anti-TER 180 ribozyme was selected for additional characterization.

To investigate whether these in vivo effects required that the anti-TER ribozyme be active, the antitumor activity of the anti-TER 180 ribozyme was directly compared with that of a ribozyme differing from it by only a single-base mutation in the catalytic core. This single-base mutation has been previously shown to abrogate ribozyme cleavage (25). Again, C57Bl/6 mice treated with cationic liposome:DNA complexes containing anti-TER 180 ribozyme showed significantly fewer metastatic lung tumors than C57Bl/6 mice treated with cationic liposome:DNA complexes containing either vector-only (P < 0.01) or the control mutant anti-TER ribozyme (P < 0.005; Fig. 3). In contrast, there was no significant difference in metastatic tumor burden of mice treated with this mutant anti-TER ribozyme compared with vector-only sequences (P > 0.1; Fig. 3).

Having demonstrated that the anti-TER 180 ribozyme produced significant antitumor effects in the B16 model system, we investigated the time course of ribozyme expression by our EBV-based expression plasmid and the kinetics of ribozyme-mediated reduction in TER expression and activity. We i.v. inoculated C57Bl/6 mice with B16-F10 melanoma cells and then with cationic liposome:DNA complexes expressing anti-TER 180 ribozyme 3 and 10 days later, as in the antitumor studies. Mice were sacrificed at various time points thereafter, and tissues were harvested for RNA expression analysis by quantitative real-time PCR. Over a time course similar to the duration of the antitumor studies, ribozyme expression levels were detectable for the duration of the experiment (on days 12–26) and were compared with levels 24 h after the second cationic liposome:DNA complex injection (day 11; Fig. 4). Ribozyme expression peaked on day 17 (1 week after the last cationic liposome:DNA complex injection), with RNA levels 8-fold higher than the day 11 time point. They were still 3–4-fold higher than on day 11 on days 20 and 21, and they decreased thereafter (Fig. 4,A). Ribozyme levels were also detectable in the heart and the spleen and were comparable between days 11 and 17 in each organ (data not shown). We also examined TER levels over this time course using the TaqMan assay. TER levels in the lung were suppressed starting on day 12 and continuing until day 21, with a maximal 23% suppression in absolute TER levels achieved on day 21 (Fig. 4 B). Hence, the EBV-based plasmid can express the anti-TER ribozyme RNA product for prolonged periods, resulting in suppression of target TER message in vivo.

Next, we examined whether systemic delivery of anti-TER 180 ribozyme can reduce the enzymatic activity of telomerase in tumor-bearing mice. The enzymatic activity of the telomerase complex in extracts of metastatic lung tumors was assayed over time after cationic liposome:DNA complex-anti-TER ribozyme injection. C57Bl/6 mice were i.v. inoculated with B16-F10 cells and subsequently treated with cationic liposome:DNA complexes containing vector-only control or anti-TER 180 ribozyme 3 and 10 days later, as in the antitumor treatment studies described above. Cationic liposome:DNA complexes containing the same control or anti-TER ribozyme inserts were re-injected into the mice at 72, 48, or 24 h before the mice were sacrificed. As shown in Fig. 5, administration of cationic liposome:DNA complexes expressing the anti-TER 180 ribozyme either 48 h (Fig. 5, Lanes 2 versus 1 and 4 versus 3) or 72 h (Fig. 5, Lane 7 versus 5 and 6) before sacrifice each reduced telomerase activity in the lung tumors by approximately 50 and 75%, respectively, when compared with vector-only sequences. No reduction was seen in the mice treated with anti-TER 180 ribozyme 24 h before sacrifice (data not shown). Thus, systemic cationic liposome:DNA complex-based anti-TER ribozyme treatments significantly suppressed in vivo TER gene function in tumor-bearing mice.

Our studies using systemic ribozyme-based targeting of TER suggest a direct link between the functional activity of the telomerase complex and the metastatic progression of B16-F10 melanoma in tumor-bearing C57Bl/6 mice. That cationic liposome:DNA complex-based ribozyme targeting was able to produce significant antitumor effects in a mouse tumor model system is of significance because of the potential challenges inherent in using such a model. Laboratory mouse strains, such as the one used here, have very long telomeres and high telomerase activity in many of its normal tissues (26). Thus, systemic targeting of telomerase in this system might have been expected to produce either widespread toxicity (if successful in every organ), or no effects given the time frame over which the level of telomerase complex was reduced.

The lack of antitumor activity of the disabled mutant anti-TER 180 ribozyme strongly suggests that the in vivo mechanism of ribozyme action is TER cleavage. We note that specific features of the systemic delivery approach used here may explain why the anti-TER 180 ribozyme could produce significant antitumor effects in vivo without widespread host toxicity. This cationic liposome:DNA complex-approach has been previously shown to preferentially transduce metastatic tumor cells and vascular endothelial cells, compared with other normal cell types (21, 22, 27). Thus, even though no ligands or promoters were used to restrict ribozyme expression in the host, this approach does not transfect all cells equally, thereby potentially limiting host toxicity despite a systemic route of administration. Interestingly, although TER levels were reduced in tumor-bearing lungs after ribozyme treatment, no significant reduction of TER gene expression was noted in the heart (data not shown), further supporting the differential targeting of this treatment approach.

Moreover, inclusion of EBV-based sequences in the plasmid vector, which has been shown to substantially prolong the duration of gene expression in vivo(20), likely contributed to the suppression of telomerase activity and of metastatic progression observed. This plasmid-based vector system has previously been shown to produce significant antitumor activity when used for delivery and expression of ribozymes targeting nuclear factor-κB, platelet endothelial cell adhesion molecule-1, or integrin β3(21). The magnitude of antitumor activity seen with anti-TER 180 ribozyme is similar to that observed with antinuclear factor-κB pathway ribozymes (21) and with positive control cDNAs such as angiostatin, p53, and CC3 (21, 22).

The ribozyme-mediated suppression of melanoma progression observed in this study was accompanied by biological evidence of decreased telomerase functional activity at metastatic sites in tumor-bearing mice. The time course study revealed significant suppression of telomerase enzymatic activity as early as 48 and 72 h after i.v., cationic liposome:DNA complex-based anti-TER180 ribozyme treatment. Lack of significant inhibition of telomerase activity 24 h after ribozyme treatment likely reflects the inherent stability of the telomerase complex (28). In contrast, suppression of telomerase activity 48 and 72 h after systemic, cationic liposome:DNA complex-based anti-TER 180 ribozyme therapy reflects the ability of the EBV-based vector to prolong ribozyme expression sufficiently to inhibit target TER activity over time (15). This observation was further supported by quantitative real-time PCR analysis, which indicated that the anti-TER ribozyme was expressed in tumor-bearing lungs for at least 16 days, resulting in suppression of target TER gene expression. After ribozyme treatment, the metastatic burden was reduced to about one-half that of the controls (Fig. 1; Fig. 2), whereas TER expression levels in the lung were decreased by about 25%. It is important to note that, due to the presence of small tumors in the lung during the early phases of the quantitative real-time PCR time course assay, RNA was isolated from the entire lung and not from individually dissected lung tumors. Thus it is likely that the levels of ribozyme expression and the magnitude of suppression of TER expression are greater in the tumor cells than in the surrounding normal cells. This is supported by a recent study that showed increased expression of delivered genes in lung tumors than in surrounding normal tissues after systemic delivery of cationic liposome:DNA complexes (29). This is also supported by the telomeric repeat amplification protocol results, in which metastatic lung tumors from late time points were harvested and shown to have up to 75% reduced telomerase activity after cationic liposome:DNA complex-based ribozyme treatment. Finally, we have previously shown that the magnitude of ribozyme-mediated suppression of target gene expression is greater in metastatic lung tumors than in normal alveolar or bronchial lining cells (21). This phenomenon appears to be due to increased phagocytosis of the complexes by tumor cells (29).

Our study also began to compare the potential utility of ribozymes targeting different components of telomerase as antitumor agents. Whereas treating mice bearing metastatic B16 tumors with ribozymes targeting several different TER cleavage sites produced antitumor activity, a similar protocol evaluating three different anti-TERT ribozymes did not reduce metastatic activity. Whether failure to see an effect of targeting the TERT component of telomerase was because of inadequate ribozyme expression or lack of TERT mRNA accessibility to our ribozymes is not known. It is possible that other ribozymes targeting different sites in either the TER RNA or TERT mRNA would result in even greater antitumor effects than those seen in this study. However, these results demonstrate that targeting the TER moiety results in a significant suppression of telomerase activity. The potential advantage of targeting TER versus TERT using a ribozyme may reflect the favorable ribozyme kinetics in the case of TER, given that no protein synthesis is required for biological activity of the TER component.

Recent studies have suggested RNA interference as a novel approach for gene-specific inhibition. Discovered as a mechanism of gene silencing in plants, RNA interference exists as a double-stranded RNA capable of mediating antisense-type targeting via Watson-Crick base pairing. The use of shorter (21 bp) RNA interference sequences to produce targeted gene disruption in a mammalian cell line (30) resulted in a myriad of studies examining RNA interference in different model systems, with suggestions that small interfering RNA (siRNA) are more potent than antisense/ribozyme approaches in vitro(31, 32). However, few studies have compared these modalities directly in vivo. Moreover, whereas a recent study demonstrated the activity of siRNA when delivered by hydrodynamic transfection methods (32), another study showed that even shorter siRNA sequences can induce an IFN response (33). Thus, it is becoming clear that siRNA-mediated gene silencing can also result in non-sequence-specific effects, similar to that reported for antisense oligodeoxynucleotides (34). Moreover, the delivery, specificity, and stability of double-stranded RNA species in mammals continue to represent significant challenges to the maturation of this promising technology to the clinical arena (35). Thus, the systemic liposome-based delivery of ribozymes as described in this study represents a novel and well-documented method for the identification of melanoma progression genes (such as telomerase) in mice. It will be interesting to examine the use of the EBV-based vector to express siRNA as well as ribozymes in tumor-bearing animals.

To our knowledge, this is the first demonstration of the importance of telomerase expression and activity in the progression of cancer metastasis. Metastasis is a complex process characterized by a cascade of pathways and events that ultimately lead to the death of tumor-bearing hosts. However, the development of micrometastases (≤2 mm in diameter) is not in itself a fatal event and is compatible with long-term survival (36, 37). Rather, the progression of tumor metastasis (reviewed in Ref. 38) results in the death of the majority of cancer patients. Thus, these results identify a novel functional role for telomerase activity in the progression of metastatic cancer, further documenting the importance of telomerase in the lethal metastatic phenotype.

In summary, these results show that ribozyme-specific inhibition of telomerase activity can suppress melanoma progression. Whether telomerase is acting on this end point as a result of an effect on cell proliferation only, or on other parameters that regulate metastatic progression, is currently under investigation. Recent studies in our laboratory using B16 transformants stably expressing the anti-TER 180 ribozyme indicate that ribozyme-mediated suppression of mTER is accompanied by decreased cell growth and increased apoptosis,5 consistent with prior studies of antitelomerase ribozymes (13). Finally, the studies presented here indicate the potential clinical utility of specifically targeting TER in the therapy of cancer.

Grant support: The Herschel and Diana Zackheim Endowment Fund, American Cancer Society Research Scholar Grant RSG-03-247-01-MGO (M. Kashani-Sabet), the Steven and Michelle Kirsch Foundation and CA096840 (E. Blackburn), and United States Public Health Service Grant CA 96666 from National Cancer Institute, NIH (R. Debs). S. Li was the recipient of a Damon Runyon Postdoctoral Fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article can be found at Clinical Cancer Research Online (http://clincanceres.aacrjournals.org).

Requests for reprints: Mohammed Kashani-Sabet, University of California San Francisco Cancer Center, 1600 Divisadero Street, Box 1706, San Francisco, CA 94115. Email: kashanim@derm.ucsf.edu

5

S. Bagheri, S. Li, M. Nosrati, S. Fong, R. LaPosa, D. Moore, J.E., Cleaver, R.J. Debs, E.H. Blackburn, M. Kashani-Sabet, Direct role for telomerase in the metastatic progression of melanoma, manuscript in preparation.

Fig. 1.

A, schematic representation of anti-TER ribozyme cleavage sites on murine TER. B, diagram of EBV-based expression plasmid used to express antitelomerase ribozymes. CMV, cytomegalovirus; MCS, multiple cloning site; FR, family of repeats.

Fig. 1.

A, schematic representation of anti-TER ribozyme cleavage sites on murine TER. B, diagram of EBV-based expression plasmid used to express antitelomerase ribozymes. CMV, cytomegalovirus; MCS, multiple cloning site; FR, family of repeats.

Close modal
Fig. 2.

Analysis of total tumor counts in the lungs of mice treated with cationic liposome:DNA complexes containing various anti-TER ribozymes or vector only control. Mice were treated as described in “Materials and Methods” and sacrificed on day 28.

Fig. 2.

Analysis of total tumor counts in the lungs of mice treated with cationic liposome:DNA complexes containing various anti-TER ribozymes or vector only control. Mice were treated as described in “Materials and Methods” and sacrificed on day 28.

Close modal
Fig. 3.

Comparison of total lung tumor counts between mice treated with cationic liposome:DNA complexes containing vector-only control, anti-TER 180 ribozyme, or anti-TER 180 mutant ribozyme. Mice were treated as described in “Materials and Methods” and sacrificed on day 27.

Fig. 3.

Comparison of total lung tumor counts between mice treated with cationic liposome:DNA complexes containing vector-only control, anti-TER 180 ribozyme, or anti-TER 180 mutant ribozyme. Mice were treated as described in “Materials and Methods” and sacrificed on day 27.

Close modal
Fig. 4.

Ribozyme (A) and TER (B) expression levels in total cellular RNA from lungs of tumor-bearing mice by TaqMan analysis after cationic liposome:DNA complex-based delivery of DNA encoding anti-TER 180 ribozyme on experiment days 3 and 10 and sacrificing mice on experiment days 11–26, as described in “Materials and Methods.” Because the ribozyme is not expressed in control mice, expression is shown relative to a time point with a baseline level of ribozyme expression and normalized to a housekeeping standard gene. In A, each time point reflects relative ribozyme expression levels on that day compared with 24 h after the second i.v. cationic liposome:DNA complex injection (experiment day 11), as normalized to Gus or histone control gene expression levels. In B, TER expression in ribozyme-treated mouse lungs, as normalized to histone expression levels, was calculated relative to that of vector-only control-treated mouse lungs.

Fig. 4.

Ribozyme (A) and TER (B) expression levels in total cellular RNA from lungs of tumor-bearing mice by TaqMan analysis after cationic liposome:DNA complex-based delivery of DNA encoding anti-TER 180 ribozyme on experiment days 3 and 10 and sacrificing mice on experiment days 11–26, as described in “Materials and Methods.” Because the ribozyme is not expressed in control mice, expression is shown relative to a time point with a baseline level of ribozyme expression and normalized to a housekeeping standard gene. In A, each time point reflects relative ribozyme expression levels on that day compared with 24 h after the second i.v. cationic liposome:DNA complex injection (experiment day 11), as normalized to Gus or histone control gene expression levels. In B, TER expression in ribozyme-treated mouse lungs, as normalized to histone expression levels, was calculated relative to that of vector-only control-treated mouse lungs.

Close modal
Fig. 5.

Analysis of telomerase activity in metastatic lung tumors by telomeric repeat amplification protocol at 48 and 72 h after treatment with cationic liposome:DNA complexes containing vector-only control or anti-TER 180 ribozyme: Lane 1, 48 h post vector therapy; Lane 2, 48 h post anti-TER 180 ribozyme therapy; Lane 3, 48 h post vector therapy with higher, more efficient lipid:DNA ratio; Lane 4, 48 h post anti-TER 180 ribozyme therapy with same lipid:DNA ratio as lane 3; Lanes 5 and 6 72 h post vector therapy; Lane 7, 72 h post anti-TER 180 ribozyme therapy.

Fig. 5.

Analysis of telomerase activity in metastatic lung tumors by telomeric repeat amplification protocol at 48 and 72 h after treatment with cationic liposome:DNA complexes containing vector-only control or anti-TER 180 ribozyme: Lane 1, 48 h post vector therapy; Lane 2, 48 h post anti-TER 180 ribozyme therapy; Lane 3, 48 h post vector therapy with higher, more efficient lipid:DNA ratio; Lane 4, 48 h post anti-TER 180 ribozyme therapy with same lipid:DNA ratio as lane 3; Lanes 5 and 6 72 h post vector therapy; Lane 7, 72 h post anti-TER 180 ribozyme therapy.

Close modal

We thank Rosie Casella for preparation of the manuscript.

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